Although the overall pathway of saturated fatty acid biosynthesis is similar in all organisms, the fatty acid synthase (FAS) systems vary considerably with respect to their structural organization. Thus in Type I FAS systems, found in vertebrates and yeasts, the necessary enzymes required for fatty acid synthesis are present on one or two polypeptide chains respectively. In contrast, in Type II systems found in most bacteria and plants, each step in the pathway is catalysed by a separate mono-functional enzyme. It would therefore appear that significant selectivity of inhibition of the bacterial and mammalian enzymes is possible.
Fab I (previously designated EnvM) functions as an enoyl-acyl carrier protein (ACP) reductase (Bergler, et al, (1994), J. Biol. Chem. 269, 5493–5496) in the final step of the four reactions involved in each cycle of bacterial fatty acid biosynthesis.
The first step is catalysed by β-ketoacyl-ACP synthase, which condenses malonyl-ACP with acetyl-CoA (FabH, synthase III). In subsequent rounds malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and II respectively).
The second step in the elongation cycle is ketoester reduction by NADPH-dependent β-ketoacyl-ACP reductase (FabG). Subsequent dehydration by β-hydroxyacyl-ACP dehydrase (either FabA or FabZ) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by NADH-dependent enoyl-ACP reductase (Fab I). Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP (16C) where upon the cycle is stopped largely due to feedback inhibition of Fab I by palmitoyl-ACP (Heath, et al, (1996), J. Biol. Chem. 271, 1833–1836). Fab I is therefore a major biosynthetic enzyme which is also a key regulatory point in the overall synthetic pathway.
Early data suggested that there were two enoyl-ACP reductases in E. coli, one NADPH dependent and the other NADH dependent. However, more recent work has found no evidence for the NADPH dependent enzyme and Fab I is the only enoyl ACP reductase identified in E. coli. (Heath, et al, (1995), J. Biol. Chem. 270, 26538–26542; Bergler, et al, (1994), J. Biol. Chem. 269, 5493–5496).
It has been shown that diazaborine antibiotics inhibit fatty acid, phospholipid and lipopolysaccharide (LPS) biosynthesis and it has also been shown that the antibacterial target of these compounds is Fab I. For example derivative 2b18 from Grassberger, et al (1984) J. Med Chem 27 947–953 has been shown to be a non-competitive inhibitor of Fab I having a Ki=0.2 mM (Bergler, et al, (1994), J. Biol. Chem. 269, 5493–5496). The antibacterial activity of diazaborine derivatives against Gram-negatives and Gram positive organisms is well documented (Grassberger et al., J Med. Chem. 1984 27, 947–953; Gronowitz et al., Acta Pharm Suecica, 1971 8 377; Wersch et al U.S. Pat. No. 2,533,918; Lam et al., J. Antimirob. Chemother. 1987 20 37–45).
Conditionally lethal Fab I mutants have been constructed in E. coli and the Fab I gene from Salmonella typhimurium complements this mutation. In addition, plasmids containing the Fab I gene from diazaborine resistant S. typhimurium conferred diazaborine resistance in E. coli (Turnowsky, et al, (1989), J. Bacteriol., 171, 6555–6565) confirming Fab I as the antibacterial target of diazaborines.
Inhibition of Fab I either by diazaborine or by raising the temperature in an Fab I temperature sensitive mutant to non-permissive conditions is lethal, thus demonstrating that Fab I is essential to the survival of the organism (Bergler, et al, (1994), J. Biol. Chem. 269, 5493–5496). Laboratory generated point mutations in the Fab I gene lead to diazaborine resistant E. coli. 
Fab I is conserved in Gram negative organisms with 98% identity between E. coli and S. typhimurium Fab I (Bergler, et al, (1992), J. Gen. Microbiol. 138, 2093–2100) and 75% identity between these proteins and H. influenzae Fab I. Staphylococcus aureus FAB I of the invention shows 54% similarity to the mycobacterial protein, InhA, which is highly conserved throughout mycobacteria including M. tuberculosis. E. coli Fab I was found to be 34% identical, 57% similar to Brassica napus (rape seed) enoyl-ACP reductase and S. aureus FAB I of the present invention was also 34% identical, 57% similar. Moreover, FAB I of the present invention was found to be 44% identical, 64% similar over 252 amino acids to E. coli Fab I. FAB I of the present invention is only 27% identical, 48% similar to a mammalian 2,4-dienoyl-coenzyme A reductase. This mammalian homolog differs from FAB I in that it is involved in the β-oxidation of polyunsaturated enoyl-CoAs and utilizes NADPH as cofactor rather than NADH. Therefore, there is significant potential for selective inhibition of FABI. Since there are no marketed antibiotics targeted against fatty acid biosynthesis it is likely that inhibitors of FAB I will not be susceptible to current antibiotic resistance mechanisms. Moreover, this is a potentially broad spectrum target.
There is an unmet need for developing new classes of antibiotic compounds. Clearly, there is also a need for factors, such as FABI, that may be used to screen compounds for antibiotic activity, such as a simple high through-put assay for screening inhibitors of FAS. Such factors may also be used to determine their roles in pathogenesis of infection, dysfunction and disease. Identification and characterization of such factors, which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases are critical steps in making important discoveries to improve human health.